Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-09-09 - 2014-07-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE Experimental Toxicology and Ecology
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Dipropylene triamine
- Physical state: liquid, colorless, clear
- Analytical purity: 99.347 area-%
- Test substance No.: 05/0696-3
- Batch No.: 00002877L0
- Date of production: 25 Feb 2013
- Stability under test conditions: guaranteed until 25 Feb 2015
- Storage condition of test material: room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: 28.73 g (mean body weight)(Exp.1); 27.66 g (mean body weight)(Exp.2)
- Assigned to test groups randomly: yes, randomization plan prepared with an appropriate computer program
- Housing: single housing in Makrolon cages, type M II cages
- Diet: Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland); ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Fully air-conditioned rooms with central air conditioning
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: due to the good solubility of the test substance
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The substance was dissolved in deionized water
- To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.
- All test substance formulations were prepared immediately before administration.
Duration of treatment / exposure:
single administration with a volume of 10 mL/kg body weight of the test substance preparation, positive control or vehicle
Frequency of treatment:
once orally
Post exposure period:
Depending on the test group in the 1st Experiment 24 and 48 hours and in the 2nd Experiment 48 and 72 hours after test substance administration, the animals were sacrificed and both femora each were excised for the preparation of the bone marrow smears.
Doses / concentrations
Remarks:
Doses / Concentrations:
150, 300 and 600 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5 male animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control No. 1: 20 mg/kg bw cyclophosphamide (CPP)
Positive control No. 2: 0.15 mg/kg bw vincristine sulfate (VCR)

The positive controls, both dissolved in deionized water, were administered to animals once orally (cyclophosphamide, CPP) or intraperitoneally (vincristine sulfate, VCR) each in a volume of 10 mL/kg body weight.
The stability of CPP and VCR is well-defined under the selected conditions, since both substances are well-established reference clastogens and aneugens, respectively.

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pretest to determine the acute oral toxicity in males and females, deaths were observed at 1 000 mg/kg body weight. At 500 mg/kg, all animals
survived but weak signs of toxicity were observed. However, there were no distinct differences in clinical observations between male and female animals. Thus, only male animals were used for the cytogenetic investigations as requested by the current OECD Guideline 474.
Based on the data of the pretest a dose of 600 mg/kg body weight was defined as MTD (maximum tolerated dose) and was selected as the highest
dose in the present cytogenetic study. 300 mg/kg and 150 mg/kg body weight were administered as further doses.
TREATMENT AND SAMPLING TIMES:
In the 1st Experiment, the animals were treated once orally (gavage) with a volume of 10 mL/kg body weight of the test substance and the vehicle.
The positive controls, both dissolved in deionized water, were administered to male animals once orally (CPP) or intraperitoneally (VCR) each in a
volume of 10 mL/kg body weight. The animals were sacrificed 24 hours (all test substance concentrations, vehicle, both positive controls) and 48hours (highest test substance concentration, vehicle) after the treatment, respectively.
In the 2nd Experiment, the animals were treated once orally (gavage) with a volume of 10 mL/kg body weight of the test substance and the vehicle.he animals were sacrificed 48 hours (all test substance concentrations, vehicle) and 72 hours (highest test substance concentration, vehicle)
after the treatment, respectively. With regard to animal welfare in the 2nd Experiment only one positive control, dissolved in deionized water, was
administered to male animals once orally (CPP) with a volume of 10 mL/kg body weight. The animals of this dose group were sacrificed 24 hours
after administration because experiences on the genotoxic potency of CPP after prolonged 48 hours exposure were missing in our laboratory.
The positive control animals were sacrificed in parallel to all dose groups prepared at 48 hours sacrifice interval.
DETAILS OF SLIDE PREPARATION:
- The slides were stained with eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes
- After briefly rinsing in deionized water, the preparations were soaked in deionized water for about 2 - 3 minutes
- Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL deionized water) for about 15 minutes
- After rinsing twice in deionized water and clarifying in xylene, the preparations were mounted in Corbit-Balsam

Evaluation criteria:
The test is considered valid if:
• The quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 2 000 PCEs per animal and a clear differentiation between PCEs and NCEs.
• The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
• The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data for PCEs.
• The administered positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

A finding is considered positive if:
• Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.

A test substance is considered negative if:
• The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.
Statistics:
Used program system: MUKERN (BASF SE)
The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether
there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate
in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results
Sex:
male
Genotoxicity:
positive
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Exp.1 Summary Table: Induction of micronuclei in bone marrow cells

 

Sacrifice

Interval

[hrs]

Animal

No.

PCE with Micronuclei

PCEs per

2 000

erythrocytesc

totala

[‰]

largeb

[‰]

Vehicle control

deionized water

24

5

1.0

0.0

1365

Test substance

150 mg/kg bw.

24

5

2.2**

0.1

1370

Test substance

300 mg/kg bw.

24

5

2.4

0.0

1373

Test substance

600 mg/kg bw.

24

5+

2.2**

0.1

1407

Positive control cyclophosphamide

20 mg/kg bw.

24

5

15.1**

0.1

1417

Positive control

vincristine sulfate

0.15 mg/kg bw.

24

5

44.2**

10.2**

1306

Vehicle control

deionized water

48

5

1.4

0.0

1398

Test substance

600 mg/kg bw.

48

5+

4.3**

0.0

1350

Exp. 2 Summary Table: Induction of micronuclei in bone marrow cells

 

Sacrifice

Interval

[hrs]

Animal

No.

Micronuclei in PCE

PCEs per

2 000

erythrocytesc

totala

[‰]

large MNb

[‰]

Vehicle control

deionized water

24

5

1.3

0.0

1315

Test substance

150 mg/kg bw.

24

5

1.1

0.0

1270

Test substance

300 mg/kg bw.

24

5

4.2**

0.0

1295

Test substance

600 mg/kg bw.

24

5

6.0**

0.1

1199

Positive control cyclophosphamide

20 mg/kg bw.

24

5

18.4**

0.1

1279

Vehicle control

deionized water

48

5

3.0

0.2

1322

Test substance

600 mg/kg bw.

48

5

3.5

0.5

908

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

bw. = body weight

a = sum of small and large micronuclei

b = large micronuclei (indication for spindle poison effect)

c = calculated number of PCEs per 2 000 erythrocytes (PCE + NCE) when scoring a sample of up to 10 000 PCE per test group

+ = sample of evalutation was increased up to 4 000 erythrocytes (PCE + NCE) per animal to confirm the data

* = p ≤ 0.05

** = p ≤ 0.01

Clinical observations1st and 2nd Experiment:

- Dose: 150 mg/kg bw: no clinical signs

- Dose: 300 mg/kg bw: no clinical signs

- Dose: 600 mg/kg bw (24 hrs): all animals showed piloerection 1h, 2h ,4h and 1d after test substance application; hunched posture was observed 1h, 2h, and 4h after test substanc application in all animals

- Dose: 600 mg/kg bw (48 hrs): all animals showed piloerection 1h, 2h, 4h and 1d after test substance application; hunched posture was observed 1h, 2h and 4h after test substance application in all animals. No clinical signs 2d after substance application .

-Dose: 600 mg/kg (72 hrs- only 2nd Experiment): all animals showed piloerection 1h, 2h, 4h and 1d after test substance application; hunched posture was observed 1h, 2h, 4h after test substance application in all animals. No clinicals signs 2d and 3d after substance application.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): positive
Executive summary:

Thus, under the experimental conditions chosen, the test substance Dipropylene triamine showed the potency to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in bone marrow cells of NMRI mice in vivo at delayed 48-hour and 72-hour sacrifice interval.